To determine the functional significance of endoplasmic reticulum chaperones in hematopoietic cells, we analyzed the expression and post-translational modification of BiP/GRP78 and GRP94 as well as the cytoplasmic chaperones HSP70 and HSC70 during the differentiation of a mouse myeloid leukemia cell line, M1. The amounts of BiP/GRP78 and GRP94 increased several-fold when M1 cells were induced to differentiate into macrophage-like cells by treatment with interleukin-6 (IL-6). Synthesis began to increase at 4 hr after IL-6 treatment. The phosphorylated form of BiP/GRP78 increased during the later stages of differentiation. These data suggested that the chaperone activity of BiP/GRP78 and GRP94 may be needed for differentiated macrophage-like cells or for the differentiation event itself, and that functionally different BiP/GRP78 accumulate during the differentiation of M1 cells.